Authors
Creative Biogene
Summary
Creative Biogene is a leading biotechnology company offering the best Lactic acid bacteria genome editing services. With years of experience and expertise in microbial genome editing, our talented scientists will work closely with you to provide any help in Lactic acid bacteria genome editing services.
Lactic acid bacteria (LAB) is a functionally related group of bacteria comprising the families Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae, which are phylogenetically diverse. They have a highly fermentative lifestyle, converting a range of sugars into mainly lactic acid. LAB play an important role in different forms of food-related biotechnology. They are gaining attention towards novel uses due to their safety for human and animal consumption, metabolic versatility and wide ecological niche adaptation (including industrial-scale fermentations).
Introduction
Lactic acid bacteria (LAB) Genome Editing Based on Homologous Recombination
Red/ET Recombination permits the engineering of DNA in LAB using homologous recombination mediated by phage protein pairs, either RecE/RecT or Reda/Redb. The central step in Red/ET recombination is the crossover step between a targeting construct containing homology arms and the target which can be a gene locus on the LAB chromosome by designing a homologous fusion fragment of the target gene, it is cloned into a suicide vector, and the suicide vector is transformed into the target LAB. An insertion mutant is selected by antibiotic screening. Under the second round of reverse selection pressure, only the mutation that contain second homologous recombination and the loss of the suicide plasmid can survive. By PCR screening and sequencing, we can obtain the mutant of the strain.
CRISPR/Cas9-mediated LAB Genome Editing
CRISPR technology, which derived from the immune system present in bacteria and archaea, is an efficient genome-scale editing tool that has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering. It enables fast and reliable genetic manipulation in LAB. Two components are requested to work: a guide RNA (gRNA), e.g. under an RNA polymerase III promoter, and the nuclear localization tag fused DNA endonuclease, with Cas9 being the most commonly used.
Here we use CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) to accomplish the LAB genome editing.
When Cas9 protein and gRNA are expressed in bacteria cells, Cas9 introduces DSBs that must be repaired by the cells via non-homologous end joining (NHEJ) or homologous recombination (HR). By supplying a DNA repair template for use in HR, various DNA modifications can be obtained.
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